The ribosome is the central component of an extremely accurate cellular protein synthetic apparatus. Its job is to rapidly and accurately decode mRNAs by reading three base "codons." With the advent of molecular genetics, it has been possible to create and examine the effects of mutants of individual ribosomal components on different ribosome-associated functions using specialized assay systems. The application of these new tools to classic biochemical methods are leading to a deeper understanding of the roles that many of the ribosomal proteins and ribosomal RNAs (rRNAs) play in determining how ribosomes maintain translational reading frame. It is now clear that the rRNAs are the central players in the reactions catalyzed by ribosomes, and that the individual rRNAs are actively involved in different ribosome functions. However, although it is highly conserved throughout evolution, the precise function of the ubiquitous 5S rRNA remains undetermined. In the past, the major barrier to studies of 5S rRNA was the fact that eukaryotic cells harbor multiple chromosomal copies of the 5S rDNA genes, which precluded any genetic dissection of 5S rRNA function, or structural studies within the context of the ribosome. We have overcome this hurdle by constructing a strain in which all 5S rRNAs are expressed from plasmid-borne clones. A global mutagenesis study of 5S rRNA using this system has revealed novel phenotypes indicative of new functions for 5S rRNA, and we are now poised to link functional aspects of 5S rRNA to its structure within the ribosome. To this end, we have enlisted the aid of Dr. Olga A. Dontsova, chief of one of the world's premier rRNA structural laboratories. The proposed collaboration will exploit the genetic and biochemical strengths of the Dinman laboratory with the proven molecular and biochemical expertise of Dr. Dontsova' s group. The broad aim of this proposal is to determine the effects of mutations in 5S rRNA on its own topology, and on the structures of the other major rRNAs. The project is designed to build up, from the effects of these mutants on the structure of 5S rRNA alone, through their effects on its association with ribosomal protein L5, and then to studies on intact ribosomal 60S subunits and whole ribosomes. This research will be done primarily in Russia as an extension of NIH grant # R01-GM62143. The information gleaned from these studies will represent a tremendous expansion of our knowledge of eukaryotic rRNA structural interactions, and will provide the scientific community with an entirely new understanding of how 5S rRNA helps to ensure that ribosomes accurately translate an organisms' genetic information into proteins.